JPH031958B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to hybrid cell lines for the production of monoclonal antibodies against theophyllin (1,3-dimethylxanthine), homogeneous and monospecific antibodies, and their use in immunoassays against theophyllin. . In 1975, Kohler and Milstein established a continuous hybrid cell line (hybridoma) derived from the fusion of mouse bone marrow cells to immunized mouse spleen cells that secreted monoclonal antibodies against sheep red blood cells. Reported [“Nature” No.
Volume 256, page 495 (1975)]. Since then, numerous papers have been submitted for the production of monoclonal antibodies against other antigens and haptens. For example, F.
“Current Topics in Microbiology and
Immunology, Volume 81 (published in 1978) and references cited therein, as well as Monoclona Autibodies, edited by R. Kennet, T. McKearn, and K. Bechtol et al.
(published in 1980) and references cited therein. Although the general techniques for producing hybridomas are well known and understood, there are still significant difficulties in preparing and selecting hybridoma cell lines that secrete antibodies with a desired set of properties. European Patent Application No. 25722 (published on March 25, 1981)
discloses the production of monoclonal antibodies against human T-mycobacterial cell surface antigens. The production of monoclonal antibodies against the cardiac glycoside digoxin was
Proceedings” Vol. 39, p. 928 (1980) and “Scand.J.Clin.Lab.Invest.” Vol. 4, p. 75 (1981)
It was reported in 2008). There is a rapidly increasing need for clinical diagnostic quantitative methods that can be used to monitor the levels of various therapeutic agents in body fluids. Theophylline, an asthma treatment, has a very narrow range of effectiveness. Immunoassay for theophylline requires highly specific antibodies. This is because other xanthine compounds that are structurally similar to theophylline exist in body fluids, and if they are recognized by the anti-theophylline antibody, they will result in a false value of the theophylline concentration being analyzed. Theophylline is 1,3-dimethylxanthine, and the four most frequently encountered xanthine compounds with cross-reactivity are caffein (1,3,7-trimethylxanthine), theobromine (3,7-dimethylxanthine), xanthine and hypoxanthine. The most frequently occurring xanthine compound is caffein, which is ubiquitous in everyday beverages. It is also a product of theophylline metabolism in newborns, and it is possible that the levels of caffein approach those of theophylline.
Therefore, it is essential that anti-theophylline antibodies used in diagnostic immunoassays for theophylline do not cross-react with caffein. Theophylline and other compounds, which generally have a formula weight of less than 1000, do not act as immunogens unless they are bound to a carrier that is itself immunogenic (see, for example, "Immunology" by HNEisen, Harper & Row). )
See 1980 edition]. Such compounds are called haptens, and various methods are known in the art for attaching haptens to carriers for immunogenicity. It is known that the choice of carrier and the site at which the hapten is attached to the carrier influences the immunogenic properties of the hapten-carrier complex as well as the specificity of the antibodies produced [e.g. Methods in Enzymology, Vol. 70]
See Volume 85 (1980)]. US Pat. No. 4,156,081 describes the synthesis of 3-position substituted theophylline derivatives and their use as immunogens to produce antibodies that do not cross-react with caffein. However, they cross-react with 1-methylxanthines.
Furthermore, commercial immunoassays require large quantities of antiserum, the production of antibodies by immunization of animals is slow and laborious, and the breeding strain varies between animal species and even within the same species. reproducibility is not necessarily good. European Patent Application No. 44441 (published on January 27, 1982)
discloses the production of monoclonal antibodies against drugs. However, it does not disclose a monoclonal antibody to theophylline that lacks any cross-reactivity to caffein. The cell lines of the present invention are three consecutively hybridized monoclonal cell lines, each having the ability to produce a novel monoclonal antibody against theophylline. The cell line is a cross between mouse myeloma cells and spleen cells from mice immunized with an 8-substituted theophylline-carrier complex (immunogen). Each of the antibodies according to the invention comprises:
When measured by the particle-facilitated turbidity inhibition immunoassay method for theophylline (see U.S. Patent Application No. 315,922 (dated October 28, 1981) and Japanese Patent Application Publication No. 58-47258), the cross-reactivity with caffeine was 5.
% or less. The use of these antibodies in immunoassays for theophylline, particularly particle-facilitated turbidity inhibition immunoassays, is also anticipated. Figures 1, 2, and 3 show particle-facilitated turbidity inhibition immunoassay using monoclonal antibodies against theophylline produced by cell lines 30/15, 17/14, and 61/7, respectively. This is a standard calibration curve obtained by Monoclonal antibodies are produced by fusing spleen cells from mice immunized with the desired antigen or hapten (theophylline in the present case) to murine myeloma cells. When the compound to be targeted by a monoclonal antibody is a hapten, it is first necessary to conjugate the hapten with a high molecular weight carrier in order to obtain immunogenicity. Such carriers include proteins, polysaccharides, and various latex particles.
For the purpose of the present invention, theophylline is
The amino group of Jinkasaga hemocyanine (KLH) is bonded via the position using carbodiimide as a bonding agent, and theophylline-8-KLH is obtained. In implementation, immunization is performed several times at intervals over a period of several weeks to several months. Animals such as immunized mice are taken at each injection and the serum is tested for the presence of the desired antibodies. Any suitable assay method may be used, but
Radioimmunoassay and enzyme-linked immunosorbent assays are often used. If antibodies are detected in the serum, the animal is sacrificed and the spleen is aseptically removed for cell fusion procedures. Several mouse myeloma cell lines are known to be useful as fusion partners. The characteristics of these cell lines are described in the above-mentioned literature, namely “Current Topics in Microbiology and
Immunology”. Generally, it is preferred to choose a myeloma cell line that does not secrete its own immunoglobulin products. Fusion operations are most often performed using polyethylene glycol as the fusion promoter. Fusion promoters such as Sendai virus may also be used. The ratio of spleen cells to myeloma cells varies, but a ratio of 5 to 10:1 is most commonly used. After fusion, cells were diluted and hypoxanthine/
Pour into selective medium such as aminopritene/thymidine (HAT) medium. Spleen cells that do not fuse die after a finite number of divisions. Also, myeloma cells cannot survive in HAT medium and die due to mutations in their hypoxanthine-guanine phosphoribosyltransferase (HGPRT) gene. Suspensions of hybrids and unfused cells are diluted before culturing to limit the number of cells per vessel. Usually, dilute to 1 to 5 cells per well of a microtiter plate. Once cell proliferation is observed, the culture supernatant is assayed for the presence or absence of the desired antibody by solid phase radioimmunoassay or other suitable assay method. Cells from pits that are found to be producing the antibody of interest are harvested as clones in soft agar, or dilution is limited to ensure monoclonality. Large amounts of antibodies can be obtained by culturing hybridomas in containers and harvesting the culture supernatant, or by injecting hybridomas into mice. Antibodies are obtained from the ascites fluid by intraperitoneal injection of the hybridoma into mice of the same strain or species that have been pretreated with pristane. Antibodies are obtained from serum by intravenous injection of hybridomas. The amount of antibody per ml varies, and is usually lowest in culture supernatant and highest in ascites fluid. The monoclonal antibodies thus created are characterized by their immunoglobulin class and subclass and by their isoelectric focusing pattern. Affinity constants are obtained and antibodies are characterized based on their reactivity with a set of related antigens (haptens). The present invention consists of monoclonal antibodies against theophylline. Three different clonotypes, namely 30/15, 17/14, and 61/7, are Balb/c
A strain of mice was induced by immunization with theophylline-8-LKH. All three are 1
It is the product of the fusion of times. All three have the same weight
Although they belong to the groups of (H) and light (L) chains, their isoelectric focusing patterns are markedly different. All three bind theophylline and can be used in immunoassays to measure theophylline content. One of the immunoassays used is particle-facilitated turbidity inhibition immunoassay. None of these clonotypes cross-reacts with caffein, which is a major hindrance to immunoassays for theophylline. It also has low cross-reactivity with other xanthine compounds. Three types of hybridoma cell lines namely 30/
15, 17/14, and 61/7 were deposited with the American Strain Collection Center (ATCC) on August 5, 1982, and are HB8152, HB8153, and HB8154, respectively.
Has been given an ATCC preservation number. The hybridoma cell line of the present invention is a cross between the mouse myeloma cell line P3-NSI-1-Ag4-1 (referred to as NS-1) and Balb/c spleen cells.
The NS-1 cell line itself is derived from the Balb/c strain of mice and synthesizes kL (light) chains, but it is not secreted. NS-1 is obtained from the Salk Institute Cell Distribution Center (La Jolla, Calif., USA). The hybridoma cell lines of the invention are hybrids, as evidenced by the presence of gene products of both parental lines in each hybrid clone. Each cell line was shown to behave stably for at least one year in terms of the antibodies produced and reaction results in other quantitative assays. The 30/15 cell line was recloned on two occasions from frozen material and the 17/14 line was recloned once from frozen material. The 61/7 strain has never been recloned. Example 1 Generation of a monoclonal antibody theophylline cell line A. Preparation of theophylline-8-KLH 8-(3-carboxypropyl)-theophylline was prepared as described in U.S. Patent Application No. 315,922 (October 28, 1981).
It was synthesized according to the description of JP-A No. 58-47258). 15 mg of this compound was mixed with 7 mg of N-hydroxysucciniside and 2 dimethylformamide.
ml for 18 hours at 4°C. At the end of this time, 0.1M sodium carbonate at PH8.5, 15
200mg of KLH dissolved in ml was added. The reaction mixture was stirred for 18 hours at 4°C. Unreacted theophylline was removed by dialysis. B. Immunization of mice of the Balb/c strain with theophylline-8 emulsified in 0.3 ml of complete Freund's supplement.
- 300 μg of KLH (prepared according to A above) was injected. Three additional dose escalation injections were given at 21-day intervals in the same manner as above. Mice were bled 7 days after the last booster injection, and serum was assayed for circulating anti-theophylline antibodies by solid-phase radioimmunoassay using ^I125 -labeled protein A and particle-facilitated turbidity inhibition immunoassay. . Mice received a final booster injection intraperitoneally, and spleens were removed for fusion 4 days later. C Particle-Facilitated Turbidity Inhibition Immunoassay This assay was used to screen mouse serum as follows. i.e. mouse serum
5μ at pH7.8, total volume of 1ml contains theophylline.
Latex particles coated with HSA [see U.S. Patent Application No. 315,922 (October 28, 1981) and Japanese Patent Application Publication No. 58-47258] 12.5 μ, polyethylene glycol 6000 2.5% (V/V, final concentration)
of 0.15M phosphate buffer was added to the measuring vessel. The turbidity due to antibody-mediated aggregation of latex particles was measured at 340 nm using a spectrophotometer.
Measured at 37°C. Mice whose turbidity formation rate reached 0.2 absorbance units per 5μ of serum were selected and their spleens were later used for fusion. D Fusion The spleen was aseptically removed and a single cell suspension was prepared from it. NS-1 those spleen cells
The cells were fused with myeloma cells in a 5:1 ratio (cell numbers are 10 ⁸ and 10 ⁷ respectively) using 0.2 ml of 30% (V/V) polyethylene glycol in serum-free medium. The fused cells were washed in serum-free medium, suspended in 30 ml of serum-free medium, and seeded into 96-well microtiter plates containing a layer of mouse peritoneal macrophage phagocytes (approximately 50 μ per hole). HAT selection medium was added after 18 hours. After 5 days, each well was examined for hybrid cell colonies every other day. If hybrids were detected (approximately 2 weeks after fusion), their pores were marked and observation continued until a point was reached at which cell proliferation was desired to increase further. At this point, harvest the culture supernatant of the pores containing the hybridized cells and remove the cells.
The cells were transferred to a 24-well plate and expanded and cultured. After 3 weeks, the hybrid cells were cultured in HT medium (containing hypoxanthine and thymidine). At this stage, the macrophage phagocyte layer was discontinued as dead cells and debris were removed from the culture. E. Screening Anti-theophylline antibodies in the culture supernatants harvested above were screened by solid-state radioimmunoassay. The antigen used in the screening was theophylline-8-KLH mentioned above.
Theophylline-8- synthesized in the same manner as
BSA (20 moles of theophylline per mole of BSA). The second antibody was an I ¹²⁵ labeled goat anti-mouse Ig. A total of 56 positive holes were detected by radioimmunoassay. These holes were rescreened using I ¹²⁵ labeled protein A.
This reagent detects only antibodies belonging to the IgG class. 56 positive culture supernatant cell lines 30/15, 17/
28, including 14 and 61/7, were proven to belong to the IgG class. These 28 culture supernatants were screened for their ability to bind free theophylline. Free theophylline (final concentration 10 μg/ml)
was incorporated into a solid state radioimmunoassay and the ability to bind free theophylline was demonstrated by the reduction of antibodies of the invention bound to immobilized theophylline-8-BSA antigen. Table 1 shows the results of these quantitative assays for antibodies produced by the three cell lines. As seen in the data, the binding of these antibodies to immobilized theophylline-8-BSA is inhibited by free theophylline, but not by five times the amount of free caffein.

[Table] F Cloning by semi-limital dilution Cell lines of interest (30/15, 17/14,
61/7) was expanded to semi-critical dilution, a number that allowed cloning at approximately 3 cells per microtiter well. A certain amount of cells were also cryopreserved alive in liquid nitrogen as a safeguard against loss. A peritoneal macrophage phagocyte layer was again used. If a sufficient number of cells were present in the pores, the culture supernatant was screened again using solid-phase radioimmunoassay. Holes showing positive were selected for expansion and recloning. G. Cloning at critical dilution The selected holes were cloned at critical dilution using strict Poisson statistics. In this case, one-third of the pores would be expected to show proliferation, and only one cell proliferated in each pore.
The probability that they are descendants of several hybridoma cells is very high. If a sufficient number of cells were present in the pores, the culture supernatant was tested again for the presence of monoclonal antibodies. All three cell lines continued to produce the desired antibodies. H chain composition All monoclonal antibodies derived from three cell lines, namely 30/15, 17/14, and 61/7, were assayed by agar gel double diffusion method.
It consists of gamma heavy (H) chains (first subclass) and katsupa light (L) chains. Example 2 Production of monoclonal antibodies in ascites carcinoma To produce large amounts of anti-theophyllin monoclonal antibodies, approximately 10 ⁶ hybridoma cells were injected intraperitoneally into Balb/c mice of the same strain pretreated with pristane. . The ascites fluid collected had high concentrations of anti-theophyllin activity, as demonstrated by both solid-state radioimmunoassay and particle-facilitated turbidity inhibition immunoassay, as described below. Example 3 Immunoquantitative assay for theophylline Ascitic fluid from mice pretreated with cell lines 30/15, 17/14 and 61/7 was analyzed for particle-promoted turbidity in an aca ^TM clinical analyzer (DuPont product). It was used for quantitative inhibition immunoassay. Theophylline 0~
20μ of a serum-based theophylline calibration standard containing 40μg/ml is automatically injected into the analytical test pack in the filling station of the analyzer, followed by 3% (container volume) polyethylene glycol 6000, 0.1% GAFACRE−610, and
4.980 ml of buffer containing 0.15 M phosphate is injected. The pack is automatically heated to 37°C. Latex particles coated with theophylline-HSA [U.S. Patent Application No. 315,922 (dated October 28, 1981)]
and Japanese Unexamined Patent Publication No. 58-47258] 40ml and
50μ of 750mM dithioerythritol solution was added through well holes number 2 and 3 of the analyzer breaker/mixer, respectively. Anti-theophylline antibody was obtained as sterile ascites fluid (17/
(50μ, amount depending on cell line) was added through the 6th well hole of the breaker/mixer. The rate of turbidity formation was automatically measured at 340 nm approximately 39 seconds after the start of the reaction in the breaker/mixer. Figure 1 shows a typical standard curve obtained using 28μ of 30/15 ascites fluid. Figure 2 shows a typical standard curve obtained using 17μ of 17/14 ascites fluid. Figure 3 shows a typical standard curve obtained using 61/7 ascites fluid. Example 4 Specificity of cell lines 30/15, 17/14 and 61/7 for theophylline In order to precisely measure theophylline in immunoquantitative assays, it is necessary to cross the anti-theophylline antibody against other xanthine compounds present in the sample. There should be little or no reactivity. Cross-reactivity is determined by the final concentration of the substance of interest in a sample containing 10 μg/ml of theophylline.
It is defined as the percentage of measurement error that would result if present at 10 μg/ml. The table shows antibodies against caffeine, theobromine, 1,7-dimethylxanthine, 3-methylxanthine, hypoxanthine, and xanthine.
The cross-reactivity of 15, 17/14 and 61/7 is compared with two rabbit antisera (924PO213 and 904PO518). Two types of rabbit antisera are theophylline-8-
It is typical of the more than 10 rabbit antisera raised and tested against BSA. The monoclonal antibodies of the invention produced by cell lines 30/15, 17/14 and 61/7 are specific for theophylline and are superior to polyclonal rabbit antisera.

【table】 [Brief explanation of the drawing]

In the accompanying drawings, Figures 1, 2 and 3 represent cell lines 30/15, 17/14 and 61/, respectively.
Figure 2 is a standard calibration curve obtained by particle-facilitated turbidity inhibition immunoassay using a monoclonal antibody against theophylline produced by No. 7.

Claims (1)

[Scope of Claims] 1. A monoclonal antibody against theophylline having cross-reactivity with caffeine of 5% or less. 2. The monoclonal antibody according to claim 1, which has a cross-reactivity of 30% or less to theobromine and a cross-reactivity of 5% or less to 3-methylxanthine. 3. An immunoassay method for theophylline that utilizes an anti-theophyllin monoclonal antibody with cross-reactivity to caffeine of 5% or less. 4. The immunoassay method according to claim 3, which is a particle-facilitated turbidity inhibition immunoassay method.